Anti-obese compositions containing holoptelea integrifolia extracts

ABSTRACT

The present invention discloses the extracts of  Holoptelea integrifolia  or the purified fractions isolated there from and the dietary, nutraceutical and pharmaceutical compositions comprising the same or optionally in combination with one or more known anti-obesic agents useful for the purpose of inhibition, amelioration or prevention of adipogenesis and lipolysis involved diseases. The invention further discloses a method for treating or preventing obesity and adipogenesis and lipolysis involved diseases using the compositions containing the extracts of purified fractions of  Holoptelea integrifolia.

TECHNICAL FIELD OF INVENTION

The present invention relates to an extract of Holoptelea integrifolia,more specifically the purified fraction isolated therefrom havinganti-adipogenic and pro-lipolysis activities for the purpose ofinhibition, amelioration or prevention of obesity, lipid storage diseaseand hyperlipedemia, and the manufacturing process thereof. Moreparticularly, the present invention relates to the compositionscomprising biologically effective amount of the said extract as anactive alone or optionally containing one or more of the extracts ofHoodia, coffee bean, Garcinia, green tea etc., formulated along withbiologically acceptable excipients.

BACKGROUND OF THE INVENTION

Obesity is excess body weight for a particular age, sex and height as aconsequence of imbalance between energy intake and energy expenditure.The primary causes of obesity are either due to overeating, inadequateexercise or eating disorder, some genetic disorders, underlying illness(e.g., hypothyroidism), certain medications, sedentary lifestyle, a highglycemic diet (i.e., a diet that consists of meals that give high postprandial blood sugar) weight cycling (caused by repeated attempts tolose weight by dieting, eating disorders), stress and insufficientsleep.

During the past 20 years, obesity among adults has risen significantlyin the United States. The latest data from the National Center forHealth Statistics show that 30 percent of U.S. adults of 20 years of ageand older, i.e. over 60 million people, are obese. The percentage ofyoung people, who are overweight, has more than tripled since 1980. Morethan 16% of the children and teens aged 6-19 years, that is over 9million young people, are considered overweight.

Although, the US national health objectives for the year 2010 is toreduce the prevalence of obesity to less than 15% among adults, currentdata indicate that the situation is worsening rather than improving(http://www.cdc.gov/nchs/products/pubs/pubd/hestats/overweight/overwght_adult_(—)03.htm).Obesity increases the risk of many diseases and health conditions suchas hypertension, dyslipidemia (for example, high total cholesterol orhigh levels of triglycerides), type 2 diabetes, coronary heart disease,stroke, gallbladder disease, osteoarthritis, sleep disorders,respiratory problems, tumors (endometrial, breast, and colon),arteriosclerosis and heart failure.

As per World Health Organisation's latest projections, approximately 1.6billion adults (age 15+) were overweight and at least 400 million adultswere obese globally in 2005. WHO further projects that by 2015,approximately 2.3 billion adults will be overweight and more than 700million will be obese (WHO's fact sheet No. 311, September 2006,http://www.who.int/mediacentre/factsheets/fs311/en/index.html). Obesityin Europe was recognized as a serious problem, with up to 27% of men,38% of women and 3 million children are clinically obese(http://ec.europa.eu/health/ph_determinants/life_style/nutrition/green_papemutritiongp_co183_en.pdf).The obesity was not limited to developed countries, but it was rapidlybecoming a problem in developing countries as well. The number of thoseaffected, particularly children, are continuing to increase at analarming rate. Obesity is already responsible for 2-8% of health carecosts and 10-13% of deaths in different parts of Europe(http://www.euro.who.int/obesity). Recent studies have shown thatapproximately a third of variance in adult body weights result fromgenetic influences. Leptin, an adipocyte and placenta-derivedcirculating protein, regulates the magnitude of fat stores in the bodyleading to obesity. Gastrointestinal peptides, neurotransmitters andadipose tissue may also have an etiologic role in obesity. Obesity andadipose tissue expansion increase the risk of hypertension, type 2diabetes, arthritis, elevated cholesterol, cancer and serious hormonalimbalances in women, leading to sterility. Low caloric diets with orwithout exercise can help with temporary weight loss; however, diet andexercise alone have not proven successful for long-term solutions inweight management (H. G. Preuss, et al, Nutrition Research, 2004, 24,45-48). In addition, supplementation with drugs that suppress appetite,reduce food intake, reduce dietary fat absorption, increase energyexpenditure and effect nutrient partitioning or metabolism havepotential efficacy but they are unfortunately accompanied by adverseside effects (C. A. Haller and N. L. Benowitz. Adverse cardiovascularand central nervous system events associated with dietary supplementscontaining ephedra alkaloids (New England J. Medicine, 2000, 343,1833-1838). Herbal and natural products containing Gymnema extract,Garcinia extract, or carnitine are known to prevent fat accumulationthrough the inhibition of fat absorption, enhancement of fatdecomposition, and the enhancement of fat consumption by the body.

Ever since obesity has become an epidemic, the word “diet” is gatheringlot of interest from different sections of many people. Many methods forweight reduction have been introduced, which leads to disproportion ofnourishment and abnormal body metabolism.

Obesity is the culmination of many underlying mechanisms. Obesity ischaracterized as uncontrolled adipose tissue mass in the body andrecognized as the fastest growing metabolic disorder in the world. Anincrease in adipose tissue can be the result of the production of newfat cells through the process of adipogenesis and/or the deposition ofincreased amounts of cytoplasmic triglyceride or lipid droplets percell. In the adipogenesis process, proliferation of preadipocytes orprecursor fat cells needs to be followed by the differentiation of thesecells to the mature adipocyte phenotype. Increased lipid accumulation inthe mature adipocyte cells is the most important feature of obesitydisorder. Peroxisome Proliferator-Activator Receptor gamma (PPAR-γ) ispredominantly expressed in adipocytes and is a key determination factorfor adipogenesis.

Fat is stored as triglycerides form in adipose tissue. The breakdown ofthis fat in fat cells into glycerol and fatty acids is known aslipolysis. During this process, free fatty acids are released into thebloodstream and circulate throughout the body. The hormones calledepinephrine, norepinephrine, glucagon and adrenocorticotropic hormoneinduce lipolysis. These hormones trigger 7TM receptors, which activateadenylate cyclase. This results in increased production of cAMP, whichactivates protein kinase A, which subsequently activate lipases found inadipose tissue. It is known that PPAR alpha plays an important role inregulating lipolysis through the control of lipid metabolic enzymes suchlipoprotein lipase (LPL) (Beisiegel U., Proc. Natl. Acad. Sci., USA, 96,pp 13656-13661, 1999).

Inhibition of the differentiation of pre-adipocytes into matureadipocytes leads to the reduction of new adipose tissue and reduction inthe formation of fat reserves. Modulation of adipogenesis and lipolysisin humans may thus lead to reduction in the burden of obesity. Thebody's adrenergic system plays a major part in regulating energyexpenditure and lipolysis. In this process catecholamines mobilizeenergy-rich lipids by stimulating lipolysis in fat cells andthermogenesis in brown adipose tissue and skeletal muscle. Theadrenergic receptor β3 is the principal receptor mediatingcatecholamine-stimulated thermogenesis in brown adipose tissue, which inhumans is distributed about the great vessels in the thorax and abdomen(Thomas, G N, International Journal of Obesity, 545-551, 24, 2000). Theβ₃-adrenergic receptor is also important in mediating the stimulation oflipolysis by catecholamines in the white fat cells of several species,including humans. The brown adipose tissue differs from white adiposetissue in that it has large numbers of mitochondria containing aso-called uncoupling protein, which can stimulate oxidativephosphorylation and thereby increase the metabolic rate (Peter Arner,The β3-Adrenergic Receptor—A Cause and Cure of Obesity? The New EnglandJournal of Medicine, 333: p 382-383). The role of brown adipose tissueis to oxidize lipids to produce heat and rid the body of excess fat.White adipose tissue, which includes subcutaneous and visceral adiposetissue, is much more abundant. It serves to store fat, which can bemobilized by lipolysis to generate free fatty acids for use by othertissues.

Selective agonists of β₃-adrenergic receptors are potentially useful intreating obesity because they could enhance energy expenditure with fewβ₁- or β₂-adrenergic side effects. A number of β₃-adrenergic agonistshave been developed and tested experimentally. Hence the treatment withβ3-selective agonists can markedly increase energy expenditure anddecreases body weight and obesity.

There are a few therapeutic interventions based on pharmaceutical drugs,such as phentermine (Fastin, Adipex P), for weight control but thesemethods exhibit side effects like high blood pressure, headache,insomnia, irritability and nervousness. The other important drug therapyfor weight control is Xenical (Roche Pharm. Co. Ltd., Swiss), Reductil(Abbot Co. Ltd., USA). The most common side effects are gas, cramps anddiarrhea, elevated blood pressure. All these therapies are based onactive ingredients that are of synthetic origin. Effective anti-obesetherapies with satisfactory efficacy and acceptable safety have not beendeveloped so far.

More importantly, anti-obese agents of natural origin with proven safetyare of great demand to control this growing menace. It is particularlyadvantageous for inhibition, amelioration and prevention of obesity ifan anti-obesity action can be imparted to food products and beverages,which are ordinarily ingested.

Hence, presently there is great demand for development of agents forprevention, maintenance and remedy of obesity, which are safe andeffective. The major emphasis now for many organizations around theglobe has been to develop new dietary ingredients and compositionsespecially from natural origin.

Holoptelea integrifolia is a tall tree distributed through out India,but more densely in Gujarat, Madhya Pradesh, Hardwar and in theHimalayas. It belong to Ulmaceae family and is known by many localnames, but more popularly as Kanju, Papri, nemali etc. Holopteleaintegrifolia seed oil is a good source of edible oil. Its leaves arecommonly used along with a blend of other Ayurvedic herbs in herbal teapreparations for detoxification and rejuvenation. This herbal tea isalso known to clear cellulite deposits and obliterates stretch marksthat are fall out of a weight problem. Holoptelea integrifolia was shownto exhibit antiviral activity in vitro assays. It was recognized as oneof the plant species useful to control air pollution.

However, none of the prior art reported or disclosed the application ofthe extracts of Holoptelea integrifolia plant or purified fractionsdeveloped therefrom for the amelioration of adipogenesis and lipolysisor the prevention or treatment or maintenance adipogenesis and/orlipolysis mediated disorders such as obesity.

OBJECTS OF THE INVENTION

The main object of the present invention is to provide pharmaceuticaland nutraceutical compositions comprising the extracts or purifiedfraction of Holoptelea integrifolia, useful for the inhibition ofobesity, and adipogenesis and lipolysis mediated diseases.

Another object of the present invention is to provide the process forthe extraction of the dried plant parts of Holoptelea integrifolia usingwater and polar and non-polar organic solvents alone or mixture thereof.

A further object of the present invention is to provide ananti-adipogenic and pro-lipolytic composition containing the Holopteleaintegrifolia ingredient capable of reducing body weight, total serumcholesterol level, phospholipids and triglycerides.

Yet another object of the invention is to provide a process for thepreparation of a dietary, nutraceutical and pharmaceutical compositions,useful for the treatment of hyperlipedemia, obesity, lipogenic diabetesand atherosclerosis, which helps in keeping slim.

Still another object of the present invention is to provide herbalformulation(s) in combination with other anti-adipogenic plant extractsor powders useful in lowering lipids and for the treatment ofatherosclerosis.

SUMMARY OF THE INVENTION

The present invention discloses herbal anti-adipogenic and pro-lipolyticsupplement comprising a biologically effective amount of an extract orfraction derived from Holoptelea integrifolia (hereinafter referredmerely to as “Holoptelea extract”) as an active ingredient alone orcompositions thereof. The compositions disclosed by the inventioncomprises of the said Holoptelea extract and optionally in combinationwith one or more known anti-obesic extracts and powders, along withbiologically acceptable carrier or diluents.

In accordance to the present invention, dried plant parts, includingleaf, seed, trunk and roots of Holoptelea integrifolia are repeatedlyextracted with water or with polar or non polar organic solvents, aloneor in combination. The extracts are combined, filtered, concentrated andthen subjected to purification.

In one aspect, the purified extract of Holoptelea integrifoliacomprising the active ingredient is formulated into a solid, semi-solidor liquid dosage form suitable for oral and parenteral administrationalone or in combination with one or more anti-adipogenic or anti-obesicagents.

In another aspect, the purified extract of Holoptelea integrifolia areformulated into nutraceuticals and dietary supplements including foodand beverages.

The anti-adipogenic and ptolipolytic composition comprising the extractor purified fractions of Holoptelea integrifolia of the presentinvention is effective for inhibition, amelioration or prevention ofvarious diseases caused by uncontrolled adipogenesis and lipolysisthereof, for example, obesity, over weight, lipid storage disease,hyperlipedemia, atherosclerosis, thrombosis and hypercholesterolemia.

DESCRIPTION OF THE FIGURES

Figure I presents the change in mean weight gain at weekly timeintervals in the treatment groups supplemented with Holopteleaintegrifolia extract and positive control and control group of animalsduring the study of the protective effect against diet induced obesity.

Figure II presents the change in mean weight gain at weekly timeintervals in the treatment groups supplemented with Holopteleaintegrifolia extract and positive control and control group of animalsduring the study of the therapeutic effect against diet induced obesity.

DETAILED DESCRIPTION OF THE INVENTION

In the adipogenesis process, proliferation of preadipocytes needs to befollowed by the differentiation of these cells to the mature adipocytephenotype. Increased lipid accumulation in the mature adipocyte cells isthe most important feature of this maturation process.

The uncontrolled fat accumulation in the body during the metabolicprocess is predominantly driven by the following key events. 1). Overexpression and increase in the activity of protein tyrosine phosphatase1B (PTB 1B) during the differentiation process of preadipocytes tomature adipocytes, 2). Highly increased lipid accumulation in thedifferentiated mature adipocytes. 3). Over expression and increasedactivity of Peroxisome Proliferator-Activated Receptor-gamma (PPAR-γ), aligand activated nuclear receptor that acts as a lipid sensor,integrating the homeostatic control of energy, lipid, and glucosemetabolism.

The breakdown of this fat in fat cells into glycerol and fatty acids isknown as lipolysis. The body's adrenergic system plays a major part inregulating energy expenditure and lipolysis. Tightly regulated balancebetween lipid synthesis (adipogenesis or lipogenesis) and lipidmobilization (lipolysis) adjusts the fat storage level within cells.

Based on the above information, inventors of the present invention haveundertaken screening of many herbal extracts using in vitro cell basedassay and found unexpectedly that the extracts and purified fraction ofHoloptelea integrifolia exhibit potent anti-adipogenic and pro-lipolyticactivity. These in vitro results are further corroborated by in vivoexperiments on diet induced obese animals. The Holoptelea integrifoliaextracts conferred protection against weight gain in animals on fat richdiet. Supplementing the diet induced obese animals with Holopteleaintegrifolia extract inhibited weight gain compared to the placebo groupof animals.

The part of the plant to be extracted of Holoptelea integrifolia is notspecifically limited, but includes, for example, leaf, seed, trunk, androot, preferably leaf. The plant parts to be extracted also referred toas raw material, are subjected to drying, for example, sun drying, shadedrying, freeze drying and the like.

Accordingly, the process for the preparation of anti-adipogenic andpro-lipolytic herbal supplements containing Holoptelea integrifoliaextract or fraction comprises the steps of:

-   -   a) extracting the dried Holoptelea integrifolia plant parts with        the water or organic solvent or mixtures thereof;    -   b) filtering the extract through fine filters;    -   c) evaporating the said filtrate to remove solvent to obtain the        concentrated extract,    -   d) purifying the concentrate to obtain active fraction and    -   e) optionally mixing the extract or purified fraction with a        known anti-obesic agent or antioxidant or bio-enhancer in a        blender to obtain the composition.

The extraction of the plant material of the present invention ispreferably carried out while gently stirring or allowing to stand usingan extraction solvent in an amount preferably about 500 ml to 1 lit per100 g of the extracting raw material. The extracting process is repeatedat least twice. It is convenient in view of operability that theextraction temperature is in a range from room temperature to not higherthan the boiling point of the solvent under normal pressure, and theextraction time for each cycle varies depending on the extractiontemperature and the like.

The extraction solvent is selected from water or organic solventsincluding polar organic solvents and non-polar organic solvents or themixture thereof.

Examples of the polar organic solvents include, but not limited to,lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol,propanol, isopropanol, n-butanol, isobutanol, tert-butanol and the like,and ketones such as dimethyl ketone, methyl ethyl ketone, methylisobutyl ketone and the like;

Examples of the non-polar organic solvents include, but not limited to,hexane, methyl acetate, ethyl acetate, butyl acetate, diethyl ether andthe like.

The extraction method employed in the present invention is selected fromimmersion extraction, cold homogenizing extraction, heat extraction,continuous extraction, super critical extraction and the like.

The ratio of raw material to extraction solvent expressed as the ratioof dried Holoptelea integrifolia: solvent, is preferably about 1/100 to1/2 (w/v), more preferably about 1/10 to 1/5 (w/v).

The extraction time for each cycle is in the range from 15 minutes toabout 48 hours, preferably from about 30 minutes to 24 hours.

The extracts are combined and subjected to fine filtration to removeextraction residues by methods known per se such as filtration,centrifugation and the like, to obtain an extract. The solvent isremoved from the extract by a method known per se, for example, thermaldrying, freeze drying, evaporation under vacuum, freeze drying and thelike to obtain a concentrated extract. A concentrated extract or asolution obtained by dissolving the concentrate in water and/or organicsolvent which may be further purified by a method such as, for example,ultrafiltration, liquid-liquid extraction, adsorption resin treatment,size exclusion chromatography, partition chromatography, and the like.The active ingredient may be further purified by using adsorptionchromatography, partition chromatography or ion-exchange chromatographyand may be still further purified by a conventional procedure. Theextract or purified fractions can be used for the present invention.

The extract or the fractions of Holoptelea integrifolia thus obtained,according to the present invention, is useful as a adipogenesisinhibitor and lipolysis accelerator as these products has exhibitedstrong anti-adipogenic and pro-lipolytic activities in cell basedassays.

To obtain full benefit, it is preferable that the above-mentionedextract or purified substance is used as it is, or the active ingredientis formulated into a solid, semi-solid or liquid dosage form by adding aconventional biologically acceptable carrier or diluent.

Specific dosage form includes, for example, oral agents such as tablets,soft capsule, hard capsule, pills, granules, powders, emulsions,suspensions, syrups, and pellets; and parenteral agents such asinjections, drops, suppositories and the like.

The Holoptelea integrifolia extracts or fractions may be optionallycombined with one or more of known anti-adipogenic or anti-obeseextracts or powders, specifically Garcinia combogia, green tea, greencoffee bean, eucalyptus plant extract, double salt of (−)-hydroxycitricacid from Garcinia species, Gymnema sylvestre extract, Banaba extract,carnitine, Phaseolus vulgaris extract, bitter orange (Citrus aurantium)extract, Chitosan, Conjugated linoleic acid, Glucomannan (Konjac plantextract), Green coffee bean extract, Caralluma extract, Sea weedextract, Hoodia Gordonii extract, Commiphora mukul gum resin extract,Zingiber officinalis extract, Allium sativa extract, chromium (III)complexes, DHEA, 7-KetoDHEA, and the composition obtained thereof isadministered using a method described above.

The anti-adipogenic and/or pro-lipolytic compositions of Holopteleaintegrifolia extracts or fractions, further comprise effective amountsof pharmaceutically or nutritionally or dietetically acceptableantioxidant(s), adaptogen(s), anti-inflammatory agents, anti-diabeticagent, bio-protectants, bio-availability enhancers and trace metals ormixtures thereof to form a formulation.

The anti-adipogenic and pro-lipolytic formulations in the presentinvention is prepared by formulating the extracts or purified fractionsof Holoptelea integrifolia, or compositions thereof along with thebiologically acceptable carrier or diluents.

The examples of the biologically acceptable carrier or diluents employedin the present invention includes, but are not limited to, surfactants,excipients, binders, disintegrators, lubricants, preservatives,stabilizers, buffers, suspensions and drug delivery systems.

Preferred examples of solid carriers include, glucose, fructose,sucrose, maltose, sorbitol, stevioside, corn syrup, lactose, citricacid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbicacid, dl-.alpha.-tocopherol, glycerin, propylene glycol, glycerin fattyester, polyglycerin fatty ester, sucrose fatty ester, sorbitan fattyester, propylene glycol fatty ester, acacia, carrageenan, casein,gelatin, pectin, agar, vitamin B group, nicotinamide, calciumpantothenate, amino acids, calcium salts, pigments, flavors, andpreservatives.

Preferred examples of liquid carriers (diluents) include, distilledwater, saline, aqueous glucose solution, alcohol (e.g. ethanol),propylene glycol, and polyethylene glycol; and oily carriers such asvarious animal and vegetable oils, white soft paraffin, paraffin, andwax.

In alternative aspect of the invention, the product of the presentinvention is delivered in the form of controlled release tablets, usingcontrolled release polymer-based coatings by the techniques known in theart. The said formulation is designed for once a daily administration.

In accordance to the present invention, the Holoptelea integrifoliaextracts or fractions is formulated into any food and drink forms suchas solid food like chocolate or nutritional bars, semisolid food likecream or jam, or gel. Contemplation was also done to formulate theproduct of the invention into a beverage and the like, such asrefreshing beverage, coffee, tea, milk-contained beverage, lactic acidbacteria beverage, drop, candy, chewing gum, chocolate, gummy candy,yoghurt, ice cream, pudding, soft adzuki-bean jelly, jelly, cookie andthe like. These various preparations or foods and drinks are useful as ahealthy food for the treatment and/or prevention of obesity.

The method of treatment teaches that the amount of the Holopteleaintegrifolia extract or purified fraction to be administered or ingestedto mammals in the form of above-mentioned nutraceutical and dietarycompositions may not be uniform and varies depending on the nature ofthe formulation and suggested human or animal dosage of the extract orthe fractions, but preferably within a range from 0.01 to 300 mg/kgweight/day.

The quantity of the extract or the purified fraction in theabove-mentioned various foods and beverage compositions may also not beuniform and varies depending on the nature of the formulation andsuggested human or animal dosage of the extract or the fractions, forexample, about 0.0001 to 50 wt %, preferably about 0.001 to 20 wt %,more preferably about 0.01 to 10 wt %.

The supplementation of Holoptelea integrifolia extract or purifiedfraction in the composition of the present invention contains about1%-100% by weight of the above extract based on the total weight of thecomposition.

The health care food of the present invention comprises the aboveextract up to 0.1 to 80%, preferably 1 to 50% by weight based on thetotal weight of the composition.

The animal feed in the present invention is prepared by mixing theHoloptelea integrifolia extract or fractions with various componentsused in the animal feed for the purpose of inhibition, amelioration orprevention of obesity, lipid storage disease, hyperlipedemia,cardiovascular disease, artherosclerosis and thrombosis, or for thepurpose of inhibition or reduction of an amount of triglyceride or anamount of cholesterol in blood, inhibiting or preventing obesity.

The form of the food additive for animal feed is not specificallylimited and the Holoptelea integrifolia extract may be added to foodproducts as it is, or as a composition, to various cooked and processedfood products. The quantity may be the same as that used in case of foodproducts. Similarly, the ingredients may also be added during or afterpreparation of the animal feeds.

The invention also describes a method of treating obesity and,adipogenesis and lipolysis involved diseases in mammals comprisingadministering to a mammal in need a therapeutically effective amount ofHoloptelea integrifolia extract or purified fraction or compositionsthereof.

The mammal is a human or an animal. The route of administration can betopical or oral or parenteral.

The following examples, which include preferred embodiments, will serveto illustrate the practice of this invention, it being understood thatthe particulars shown are by way of example and for purpose ofillustrative discussion of preferred embodiments of the invention.

Examples Example 1

Dried leaves of the plant material Holoptelea integrifolia (1 Kg) waspulverized to coarse powder, extracted with methyl alcohol (5 L) at roomtemperature (RT) for 1 hr. Extraction process was repeated thrice usingmethyl alcohol (3 L+3 L+2 L). All the extracts were combined and thecombined alcoholic extracts were fine filtered, and the clear extractwas evaporated to dryness on a climbing film evaporator at 50-60° C.under vacuum to obtain the residue (80 g)

Example 2

Dried leaves of the plant material Holoptelea integrifolia (1 Kg) waspulverized to coarse powder, extracted with water/methyl alcohol (40:60;6 L) at RT for 1 hr. Extraction process was repeated three times usinghydroalcohol 40/60 (5 L+3 L+3 L). All the water/methyl alcohol extractswere combined, subjected to fine filtration, and the clear extract wasevaporated to dryness on a climbing film evaporator at 50-60° C. undervacuum to obtain the residue (100 g).

Example 3

Dried leaves of the plant material Holoptelea integrifolia (1 Kg) waspulverized to coarse powder, extracted with water (6 L) at RT for 1 hr.Extraction process was repeated three times using water (4 L+4 L+2 L).All the extracts were combined, the combined aqueous extracts were finefiltered, and the clear extract was evaporated to dryness on a climbingfilm evaporator at 50-60° C. under vacuum to obtain the residue (105 g).

Example 4

The Holoptelea integrifolia methanol extract (50 g) was dissolved in 30%methanol (1 L) and eluted through R-20 resin column (550 mL, syntheticadsorbent) and the column washed with water (1 L). The resin was theneluted with 80% methanol (1 L). The fractions eluted with 80% methanolwere combined and the solvent evaporated under reduced pressure to givethe bio-enriched fraction of Holoptelea integrifolia extract (23 g).

Example 5

Assessment of Inhibition of Lipid Accumulation in DifferentiatedAdipocytes by Holoptelea integrifolia Extract:

One hundred thousand 3T3-L1 Human pre-adipocyte cells in Dulbecco'sModified Eagles Medium (DMEM) containing 10% Fetal Bovine Serum (FBS)were taken into each well of a 24-well plate and incubated for 48 h at37° C. and 5% CO₂. The differentiation of pre-adipocyte cells wasinitiated in a differentiation medium containing 10 μg/ml insulin, 1.0μM dexamethasone, and 0.5 mM isobutylmethylxanthine (IBMX) for 48 h.After this the medium was replaced by DMEM containing 10 ug/ml insulinand incubated for 3 days. Then the differentiating cells were treatedwith 10 μg/ml of Holoptelea integrifolia hydroalcohol (60% methanol)extract or methanol extract or bio-enriched fraction separately andmaintained in the medium for another 3-5 days. The cells incubated with0.1% DMSO were considered as the vehicle control. After the incubationperiod, cells were washed with phosphate buffered saline (PBS) and fixedwith 10% buffered formalin for 1 h at room temperature. One smallaliquot of cell suspension was separated for cell counting inhemocytometer chamber. Fixed cells were stained with Oil Red O solutionto measure the cellular neutral lipid accumulation. Briefly, cells werewashed with PBS, fixed with 10% buffered formalin and stained with OilRed O solution (0.5 g in 100 ml isopropanol) for 10 min. After removingthe staining solution, the dye retained in the cells will be eluted intoisopropanol and OD measured at 550 nm. The inhibition of fataccumulation in the treated cells was compared with the mock treateddifferentiated adipocytes. The treated and control cells were alsoanalyzed and compared for inhibition of lipid accumulation visuallyunder microscope and recorded digitally in suitable image capturesystem. The Holoptelea integrifolia methanol and 60% methanol extractsand bio-enriched fraction showed 46%, 45% and 55% inhibition of lipidaccumulation respectively.

Example 6

Assessment of Pro-Lipolytic Activity of Holoptelea integrifolia Extractin Differentiated Adipocytes:

The lipolytic activity was assessed in mature adipocytes as per theprocedure of Chemicon International, USA, by measuring free glycerolsecreted into the culture medium. The 3T3-L1 cells were differentiatedfor 5 days and then the culture medium was removed. The monolayer waswashed twice with wash solution (Hank's balanced salt solution), andthen 250 μL of incubation solution (Hank's balanced salt solution plus2% bovine serum albumin) was added to the wells in triplicate inpresence and absence of Holoptelea integrifolia methanol extract, andthe cells were further incubated for 16 h. To measure lipolysis, 200 μLof free glycerol assay reagent was added to 25 μL of culturesupernatants and controls containing glycerol standard. The samples andthe controls were incubated for 15 min, and the absorbance was read at540 nm. A standard curve constructed from the glycerol standard was usedto calculate the concentration of free glycerol in the culturesupernatants. The percentage increase in glycerol concentration in thesample solutions compared to the control containing the knownconcentrations of glycerol corresponds to the percentage acceleration oflipolysis by Holoptelea integrifolia extracts. The percentage increasein lipolysis accelerated by Holoptelea integrifolia methanol extract is70.2%.

Example 7

Protective Effect of Holoptelea integrifolia Against Diet InducedObesity in Rats.

Selected healthy Sprague-Dawley rats were randomly assigned to controlor various treatment groups (n=6). All the animals allocated forprotective phase of the study were on dietary intervention by feedinghigh fat diet ad libitum and the animals allocated to treatment groupswere simultaneously given oral administration of 100 mg or 250 mg/kgHoloptelea integrifolia methanol extract in 10 mL of 0.5% CMC, usinggastric tube for the entire 8 week study duration. The test animals ofthe control group were simultaneously given 10 ml/kg of 0.5% CMC.Sibutramine (7 mg/kg body weight) was used as a positive control. Foodand water consumption were recorded daily, body weights were recordedweekly and fasting blood samples were collected before initiation, after4^(th) week and 8^(th) week (termination) of the study. The weight gainis plotted against the study duration in weeks (Figure I). The treatmentgroups corresponding to 100 mg/kg and 250 mg/kg doses showed 15.4% and27.3% protection respectively against weight gain, in diet inducedobesity, when compared with untreated control group. Sibutramineexhibited 30.5% reduction in weight gain compared to the control group.The animals of the treatment group also showed significant reduction inserum triglycerides and lipid profile (anti-hyperlipidimic activity).Upon administration of Holoptelea integrifolia alcohol extract for aperiod of 60 days, the level of triglyceride in the obese rats wasreduced by 50% compared to the control. The levels of serum cholesteroland LDL were reduced by 40% and 45% respectively, whereas the level ofHDL was increased by 25%.

Example 8

Anti-Obese Activity of Holoptelea integrifolia in Diet Induced ObeseRats.

Selected healthy Sprague-Dawley rats were randomly assigned to controlor various treatment groups (n=6). All the animals allocated for thestudy were made obese through dietary intervention by feeding high fatdiet ad libitum for 8 weeks. After 8 weeks, the treatment group ofanimals was given oral administration of 250 mg/kg or 500 mg/kg ofHoloptelea integrifolia alcohol extract in 10 mL of 0.5% CMC, usinggastric tube for 5 week study duration. Sibutramine (7 mg/kg bodyweight) is used as a positive control. The control group of animals weregiven 10 ml/kg of 0.5% CMC. Food and water consumption were recordeddaily, body weights were recorded weekly and fasting blood samples werecollected before initiation after 5^(th) week of the study. The weightgain for different groups compared at weekly intervals (Figure II). Thereduction in body weight gain of animals in treatment groupscorresponding to 250 mg/kg and 500 mg/kg doses are about 98.1% and130.8% respectively compared to those in the untreated control group.Sibutramine exhibited 123.1% reduction in weight gain compared to thecontrol group. The treatment group of animals also showed significantreduction in serum triglycerides and lipid profile (anti-hyperlipidemicactivity) when compared to the control group.

A review of foregoing sequence of experimental protocols shows that anextract of Holoptelea integrifolia has an excellent action of inhibitingadipogenesis, accelerating lipolysis and reducing body weight.

It will be evident to those skilled in the art that the invention is notlimited to the details of the foregoing illustrative examples and thatthe present invention may be embodied in other specific forms withoutdeparting from the essential attributes thereof, and it is thereforedesired that the present embodiments and examples be considered in allrespects as illustrative and not restrictive, reference being made tothe appended claims, rather than to the foregoing description, and allchanges which come within the meaning and range of equivalency of theclaims are therefore intended to be embraced therein.

1. Anti-adipogenic and pro-lipolytic herbal supplements for inhibitingor preventing or controlling adipogenesis and lipolysis involveddiseases in mammals, comprising an extract or purified fraction derivedfrom Holoptelea integrifolia alone or a composition comprising saidextract or fraction optionally in combination with at least oneantiobesic agent.
 2. The anti-adipogenic and pro-lipolytic herbalsupplements as claimed in claim 1, wherein the source of extract orfraction is a Holoptelea integrifolia plant part selected from thegroup, of leaf, seed, trunk, root, and mixtures thereof.
 3. Theanti-adipogenic and pro-lipolytic herbal supplements as claimed in claim1, wherein said extract or purified fraction or a composition comprisingsaid extract or fraction is used as such.
 4. The anti-adipogenic andpro-lipolytic herbal supplements as claimed in claim 1, wherein saidadipogenesis and lipolysis involved diseases comprise obesity,overweight, hyperlipedemia, type 2 diabetes, cardiovascular disease andatherosclerosis. 5-15. (canceled)
 16. The anti-adipogenic andpro-lipolytic herbal supplements as claimed in claim 1, wherein saidextract or purified fraction or a composition comprising said extract orfraction is formulated into a nutraceutical or pharmaceutical dosageform along with biologically acceptable excipients.
 17. Theanti-adipogenic and pro-lipolytic herbal supplements as claimed in claim16, wherein the dosage form is a nutraceutical or pharmaceutical dosageform suitable for oral administration, and is selected from the groupconsisting of tablets, soft capsules, hard capsules, pills, granules,powders, emulsions, suspensions, syrups, and pellets.
 18. Theanti-adipogenic and pro-lipolytic herbal supplements as claimed in claim16, wherein said nutraceutical or pharmaceutical dosage form is suitablefor parenteral administration, and is selected from the group consistingof injections, drops, and suppositories.
 19. The anti-adipogenic andpro-lipolytic herbal supplements as claimed in claim 16, wherein saidnutraceutical or pharmaceutical dosage form is in the form of a dietaryformulation selected from the group consisting of foods for specifiedhealth uses.
 20. The anti-adipogenic and pro-lipolytic herbalsupplements as claimed in claim 1, wherein the extract or purifiedfraction derived from Holoptelea integrifolia is prepared by: a)extracting dried Holoptelea integrifolia plant parts with water or anorganic solvent or a mixture thereof to produce an extract solution; b)filtering the extract solution through fine filters to produce afiltrate; c) evaporating said filtrate to remove solvent to obtain aconcentrated extract, d) purifying the concentrated extract to obtainsaid extract or purified fraction; and e) optionally mixing the extractor purified fraction with an anti-obesic agent or antioxidant orbio-enhancer in a blender.
 21. The anti-adipogenic and pro-lipolyticherbal supplements as claimed in claim 20, wherein extracting driedHoloptelea integrifolia plant parts comprises extracting the driedHoloptelea integrifolia plant parts with an organic solvent selectedfrom the group consisting of polar organic solvents selected from thegroup consisting of methanol, ethanol, propanol, isopropanol, n-butanol,isobutanol, tert-butanol, dimethyl ketone, methyl ethyl ketone, andmethyl isobutyl ketone; and non-polar organic solvents selected from thegroup consisting of methyl acetate, ethyl acetate, butyl acetate, anddiethyl ether.
 22. The anti-adipogenic and pro-lipolytic herbalsupplements as claimed in claim 1, wherein the at least one antiobesicagent is selected from the group consisting of the extracts or powdersof Garcinia cambogia, green tea, green coffee bean, eucalyptus plantextract, double salt of (−)-hydroxycitric acid derived from Gareiniaspecies, Gymnema sylvestre extract, Banaba extract, carnitine, Phaseolusvulgaris extract, bitter orange (Citrus aurantium) extract, Chitosan,Conjugated linoleic acid, Glucomannan (Konjac plant extract), Greencoffee bean extract, Caralluma extract, Hoodia Gordonii extract,Commiphora mukul gum resin extract, Zingiber officinalis extract, Alliumsativa extract, chromium (111) complexes, DHEA, and 7-Keto-DHEA.
 23. Amethod for treating or preventing obesity or hyperlipedemia in apatient, comprising: administering a herbal supplement according toclaim 1 to said patient, wherein the herbal supplement is administeredas a nutraceutical or pharmaceutical dosage form selected from the groupconsisting of a food, a beverage, an oral dosage form, or a parenteraldosage form.
 24. A method for lowering plasma triglyceride in a patient,comprising: administering a herbal supplement according to claim 1 tosaid patient, wherein the herbal supplement is administered as anutraceutical or pharmaceutical dosage form selected from the groupconsisting of a food, a beverage, an oral dosage form, or a parenteraldosage form.
 25. The anti-adipogenic and pro-lipolytic supplements asclaimed in claim 1, further comprising an effective amount of at leastone pharmaceutically or nutritionally or dietetically acceptableantioxidant, adaptogen, anti-inflammatory agent, anti-diabetic agent,bio-protectant, bioavailability enhancer, trace metal, or mixturethereof to form a formulation.
 26. The anti-adipogenic and pro-lipolyticsupplements as claimed in claim 1, wherein the extract or purifiedfraction derived from Holoptelea integrifolia is administered to amammal in the form of a therapeutic formulation or food or beverage in arange from 0.01 to 300 mg/kg weight/day.
 27. A method of treatingobesity and, adipogenesis and lipolysis involved diseases in mammalscomprising administering to a mammal in need thereof a therapeuticallyeffective amount of a Holoptelea integrifolia extract or purifiedfraction or a composition comprising said Holoptelea integrifoliaextract or purified fraction, optionally in combination with at leastone anti-obesic agent.
 28. The anti-adipogenic and pro-lipolytic herbalsupplements as claimed in claim 2, wherein said extract or purifiedfraction or a composition comprising said extract or fraction is used assuch.
 29. The anti-adipogenic and pro-lipolytic herbal supplements asclaimed in claim 2, wherein said extract or purified fraction or acomposition comprising said extract or fraction is formulated into anutraceutical or pharmaceutical dosage form along with biologicallyacceptable excipients.
 30. The anti-adipogenic and pro-lipolytic herbalsupplements as claimed in claim 16, wherein said dosage form is anutraceutical dosage form is in the form of a dietary formulationselected from the group consisting of chocolate bars; nutritional bars;creams; jams; gels; beverages selected from the group consisting ofcoffee, tea, milk-containing beverages, and lactic acidbacteria-containing beverages; candies, chewing gums, chocolates, gummycandies, yoghurt, ice cream, puddings, soft adzuki-bean jellies,jellies, and cookies.